Hybridoma technology has been widely used to isolate antibodies for a variety of applications (Kohler and Milstein, 1975, Nature, 256, 495-7). Monoclonal antibodies have not only been indispensable as reagents but have also been developed as drugs to treat various human disease conditions including cancer, autoimmune, inflammatory and cardiovascular diseases, and viral infections (Reichert and Pavlou, 2004, Nat Rev Drug Discov, 3, 383-384). While the use of hybridoma technology to generate monoclonal antibodies is common practice in today's research and development laboratories, identification of the monoclonal antibodies that possess the desired binding and functional characteristics is a labor-intense, time-consuming process. This is largely due to the fact that hybridoma cells secrete, and are therefore disassociated from, the desired antibodies, thus making it an extensive and costly process to isolate the single hybridoma clones that secrete the antibody of interest.
The need for a rapid high-throughput screen of antibodies that specifically bind a specified target led to the development of cell surface display technologies in which the antibody-producing host cell remains physically associated with the displayed antibody of interest. This allows rapid isolation and sequence identification of the gene encoding the displayed antibodies having the desired binding characteristics. Such methods for identifying monoclonal antibodies include antibody display technologies using bacteria, yeast and ribosomes (Amstutz et al., 2001, Curr Opin Biotechnol, 12, 400-405; Wittrup, 2001, Curr Opin Biotechnol, 12, 395-399).
Surface display technologies are also valuable for screening libraries of antibody fragments generated using variable domains isolated from diverse species immunized with the target antigen of interest. While the mouse is the most common source of monoclonal antibodies against human proteins, it is not always possible to raise high affinity antibodies against certain antigens or epitopes that are highly conserved between human and mouse, as such antigens have little or no immunogenicity in mice due to tolerance (Rajewsky, 1996, Nature, 381, 751-8). In such cases, immunization of non-rodent species, e.g. rabbit or chicken, is an alternative way to raise specific antibodies that bind a target of interest. DNA sequences encoding immunoglobulin variable domains are then generated from antibody-producing cells isolated from these immunized animals and cloned into a display library expression system.
In commonly used display library systems, antibodies are typically displayed as single chain Fv (scFv) or Fab fragments because the use of smaller sized fragments makes them amenable to phage display. Thus, characterization of the biological activities and further development of the isolated antibody fragments often requires conversion to whole immunoglobulins and expression in mammalian cells for proper folding and post-translational processing. This conversion process may produce antibodies with binding characteristics unlike those selected for in the initial screen.
We have developed a versatile mammalian expression vector that allows expression of membrane-bound and soluble forms of a selected immunoglobulin. The expression vectors described herein allow efficient conversion of full-length cell surface-bound immunoglobulins, which are used for the initial screening of specific binders, to the full-length secreted form of the selected binder immunoglobulin, which can be functionally characterized.